The Journal of Infectious Diseases SUPPLEMENT ARTICLE Budding of Ebola Virus Particles Requires the Rab11-Dependent Endocytic Recycling Pathway Asuka Nanbo and Yusuke Ohba Faculty of Medicine and Graduate School of Medicine, Hokkaido University, Sapporo, Japan The Ebola virus-encoded major matrix protein VP40 traffics to the plasma membrane, which leads to the formation of filamentous viral particles and subsequent viral egress. However, the cellular machineries underlying this process are not fully understood. In the present study, we have assessed the role of host endocytic recycling in Ebola virus particle formation. We found that a small GTPase Rab11, which regulates recycling of molecules among the trans-Golgi network, recycling endosomes, and the plasma membrane, was incorporated in Ebola virus-like particles. Although Rab11 predominantly localized in the perinuclear region, it distributed diffusely in the cytoplasm and partly localized in the periphery of the cells transiently expressing VP40. In contrast, Rab11 exhibited a perinuclear distribution when 2 VP40 derivatives that lack ability to traffic to the plasma membrane were expressed. Finally, expression of a dom- inant-negative form of Rab11 or knockdown of Rab11 inhibited both VP40-induced clusters at the plasma membrane and release of viral-like particles. Taken together, our findings demonstrate that Ebola virus exploits host endocytic recycling machinery to facilitate the trafficking of VP40 to the cell surface and the subsequent release of viral-like particles for its establishment of efficient viral egress. Keywords. Ebola virus; Rab11; recycling endosome; viral-like particles; virus egress; VP40. Ebola virus (EBOV), a member of the family Filoviridae, is an regulates processes that include cell motility and division, actin enveloped, single-stranded, negative-sense ribonucleic acid polymerization, and formation of filopodia [16], Sec24C, a (RNA) virus that causes severe hemorrhagic fever with a high component of the host COPII vesicular transport system [17], mortality rate in humans and nonhuman primates [1]. Currently, microtubules [18], and the HECT family E3 ubiquitin ligase there are no US Food and Drug Administration-approved ther- WWP1 [19]. However, the molecular mechanisms of VP40- apeutics to treat EBOV infection [2]. Ebola virus encodes 7 mediated EBOV egress have not been fully elucidated. structural genes that assemble to yield distinct filamentous viral The host endocytic recycling machinery and its major regula- particles. The EBOV-encoded major matrix protein VP40 traffics tor, a small GTPase Ras-related in brain (Rab) 11, are implicated to the plasma membrane (PM), which leads to the formation and in the virus lifecycle including assembly and viral egress [20]. release of the virus-like particles (VLPs), even when expressed Rab11 associates with recycling endosomes and regulates recy- alone [3–7]. VP40 has been shown to form a dimer that further cling processes of proteins and vesicles to the cell surface [21]. OA-CC-BY-NC-ND assembles into a flexible filamentous matrix structure [8]. Rab11 also localizes to the trans-Golgi network (TGN) and VP40 contains 2 overlapping late domains (PTAP and PPXY post-Golgi vesicles and has been implicated in the trafficking motifs) that interact with Tsg101, a component of the endoso- between the TGN and the endosomal recycling compartments mal sorting complex required for transport (ESCRT)-1, and through the regulated secretion pathway [22]. with the ubiquitin ligase Nedd4, respectively [9]. Although The Rab11-dependent recycling pathway involves trafficking both late domains are responsible for release of VLPs [10, 11], of virus components to their sites of egress. Results from previ- a study using recombinant EBOV showed that the late domains ous studies showed that Rab11-positive vesicles associate with are dispensable for virus replication [12]. Results from previ- viral ribonucleoproteins of a variety of viruses, including influ- ous studies showed that several host factors are required for enza A virus (IAV) [23–26], Sendai virus [27, 28], and human the intracellular transport of VP40, including actin [13–15], parainfluenza virus type I [28], and promote their trafficking IQGAP1, a ubiquitously expressed scaffolding protein that toward their sites of egress. Herpes simplex virus-1 exploits the same pathway to transport its viral glycoproteins on the PM to the intracellular compartment for maturation of virions [29]. The Correspondence: A. Nanbo, PhD, Faculty of Medicine and Graduate School of Medicine, Hokkaido University, Kita 15 Nishi 7, Kita-ku, Sapporo, Japan 060-8638 ([email protected]). Nipah virus-encoded fusion protein is activated by cleavage with The Journal of Infectious Diseases® 2018;218(S5):S388–96 cathepsin B in the recycling endosomes and subsequently recycled © The Author(s) 2018. Published by Oxford University Press for the Infectious Diseases to the PM for incorporation in the virions [30]. The accessory pro- Society of America. This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs licence (http://creativecommons.org/licenses/ tein Vpu of human immunodeficiency virus 1 is transported via by-nc-nd/4.0/), which permits non-commercial reproduction and distribution of the work, in any Rab11-positive vesicles toward the assembly site on the PM [31]. medium, provided the original work is not altered or transformed in any way, and that the work is properly cited. For commercial re-use, please contact [email protected] Moreover, a potential role for the Rab11-dependent endo- DOI: 10.1093/infdis/jiy460 cytic recycling pathway in viral egress has been suggested for S388 • JID 2018:218 (Suppl 5) • Nanbo and Ohba both positive- and negative-sense RNA viruses. The Rab11- Preparation of Ebola Virus-Like Particles dependent pathway involves budding of respiratory syncytial The preparation of Ebola VLPs was described previously [41, 42]. virus (RSV) [32, 33] and the filamentous IAV [34] by facilitat- In brief, equal amounts of the pCAGGS expression plasmids for ing fission of viral particles from the PM. The Rab11-mediated EBOV subtype Zaire, strain Mayinga VP40, glycoproteins (GP), pathway is also implicated in egress from the PM for Mason- and nucleoproteins (NP) were transfected into HEK293T cells. Pfizer monkey virus [35, 36], Jaagsiekte sheep retrovirus [37], Forty-eight hours posttransfection (h.p.t.), the culture superna- and hepatitis C virus [38], which are originally assembled and tants were harvested and centrifuged at 1500 rpm for 5 minutes mature, respectively, at the microtubule organizing center, the and then at 3500 rpm for 15 minutes to remove detached cells endoplasmic reticulum, and the Golgi/TGN compartments. and cell debris, respectively. The VLPs were pelleted through Although a previous study using liquid chromatogra- a 30% sucrose cushion by centrifugation at 11 000 rpm for 1 phy-linked tandem mass spectroscopy (LC-MS/MS) showed hour at 4°C with an SW40 rotor (Beckman, Fullerton, CA). The that Rab11b is incorporated into authentic filovirus virions [39], pelleted VLPs were resuspended in phosphate-buffered saline its physiological relevance in the EBOV lifecycle has remained (PBS). For a protease protection assay, Ebola VLPs were treated unclear. In the present study, we have assessed the role of the with or without 0.1 mg/mL trypsin in the presence or absence Rab11-dependent recycling pathway in VP40-mediated bud- of 0.05% Triton X-100 at room temperature for 30 minutes, fol- ding of Ebola VLPs. We found that Rab11 was incorporated into lowed by the addition of sodium dodecyl sulfate polyacrylamide Ebola VLPs. Although Rab11 predominantly localized in the gel electrophoresis sample buffer. The incorporation of VP40 perinuclear region, it distributed diffusely in the cytoplasm and and Rab11 in the purified VLPs was confirmed by Western partly localized in the periphery of the cells transiently express- blotting with the mouse monoclonal antibodies against VP40 ing VP40. Blocking of endosomal recycling by the expression of (clone 6; 1:4000 dilution) and Rab11 (1:1000 dilution; Becton, a dominant-negative form of Rab11 or by knockdown of endog- Dickinson and Company, Franklin Lakes, NJ), respectively. enous Rab11 suppressed the VP40-induced formation of clus- The experiment was performed 3 times independently, and the ters at the PM. Moreover, downregulation of Rab11 reduced the intensity of the bands was quantified by use of Multigauge soft- production of Ebola VLPs. Taken together, our findings indicate ware (FUJIFILM Corporation). that EBOV exploits host endocytic recycling machinery for its Immunofluorescence Staining establishment of efficient viral egress. Vero-E6 cells grown on coverslips were transfected with the expression plasmid for VP40, VP40-L117R, or VP40-I307R. At MATERIALS AND METHODS 48 h.p.t., the cells were fixed with 4% paraformaldehyde in PBS Cell Culture for 10 minutes at room temperature, permeabilized with PBS African green monkey kidney epithelial Vero-E6 cells and human containing 0.05% Triton X-100 for 10 minutes at room tem- embryonic kidney HEK293T cells (American Type Culture perature, and blocked in PBS containing 4% bovine serum albu- Collection) were grown in high-glucose Dulbecco’s modified min for 20 minutes at room temperature. The cells were then Eagle’s medium containing 10% fetal bovine serum and antibi- incubated with a mouse monoclonal antibody